CN104797268B - A kind of novel shigella attenuated live vaccine - Google Patents

Abstract

The invention discloses a kind of Shigella attenuated live vaccine, it is based on the coarse Shigella bacterial strain for lacking LPS O antigens, by the mutation for attacking plasmid, it is Non-Invasive, the immunoprophylaxis of main body is particularly used for prevent infectious diseases, it is preferred that intestines problem, an and Shigella bacterial strain, it is to delete rfbF, the Shigella flexneri 2a bacterial strains of ipaB and/or ipaC genes, and the recombinant plasmid vector based on mutation Shigella invasion and attack plasmid, the invasion and attack plasmid includes the nucleotide sequence for encoding at least one heterogenetic antigen, wherein plasmid is mutated at least one of ipaB and/or ipaC genes place.

Description

A kind of novel shigella attenuated live vaccine

The present invention relates to a kind of Shigella attenuated live vaccine, it is mutated with specific objective and produced, to cause serotype only Vertical cross protection and expressing heterologous (non-Shigella) antigen.

Background technology

Shigella is the Gram-negative enterobacteria that height is obtained from the mankind, it causes bacillary dysentery.The disease is only led to Cross directly individual contact or the pollution of the human faecal mass of food and water and propagate.Therefore, bacillary dysentery is undesirable in sanitary condition Region in it is popular.Worldwide there are 165M cases according to estimates, the death of up to 1M is most of to be occurred below five years old With children.The separated bacterial pathogen in Southeast Asia and the acute diarrhea in children event of Sub-Saharan Africa area 1-5 Sui In body, it is found that Shigella is one of most common bacterial pathogen (Kotloff et al., Bull.W.H.O.77：651- 666,1999).Bacillary dysentery is also very common in the passenger and army personnel for entering endemic disease country.People for a long time one Directly think, vaccine is vital for control dysentery, but the vaccine development of anti-Shigella is subject to serotype specificity The obstruction of immune response, i.e., exposed to Shigella (natural or vaccine mediation) when, immunoprotection is typically limited to give Serotype.Four species of Shigella include 50 serotypes and blood serum subtype altogether, according to their LPS O antigens Distinguish.

Hong et al. (Molecular Microbiology 24:779-91,1997) describe chromosome and plasmid-encoded Lipopolysaccharides gene influence of the mutation to the invasion and seroresistance of Shigella flexneri.The mutation meeting of rfb and rfaL genes Eliminate whole O antigenic side chains or produce the chain that length greatly shortens.

Nagy et al. (J.Infect.Diseases 198:1699-706,2008) describe Salmonella enteritidis and adjust fat The Vaccine Potential of more sugar mutants.The missing of the sub- RfaH of transcription antitermination causes the different LPS chains of length, " slight coarse " table Type.

Regulatory protein RfaH is shown included in Shigella flexneri (S.flexneri) long-chain (O antigen weights of i.e. high number (Carter et al.Microbiology.2007Oct in the up-regulation independently of growth period of LPS molecules again)；153(Pt 10):3499-507)。

In addition to LPS O antigens, the major virulent factor of Shigella is all unexpectedly guarded.This prompting, it is anti-to these The missing of former immunological regulation evolution pressure confirms that O antigens are the received viewpoints of unique protective antigens.But Reacted in the presence of the labelled antibody for so-called " invasion and attack plasmid antigen " (Ipa-s), particularly after the exposure repeated.These are compiled Code forms the component for the 3 type excretory systems (T3SS) be responsible for attacking and then caused a disease in the big antigen to cause a disease on plasmid.

The Structure-function analysis of Shigella virulence factor invasion and attack plasmid antigen B (ipaB) is open by Guichon et al. (J.Bacteriol.183:1269-76,2001).IpaB mutant is generated so that function and albumen subdomain to be connected.

Menard et al. (J.Bacteriol.17518：5899-5906,1993) describe the ipa genes of Shigella flexneri The mutation of ipaB, ipaC and ipaD cause the forfeiture of Shigella invasion and attack potentiality.

Non-protective is considered to be for the antibody of Ipa albumen (such as to secondary conservative antigen), because should not Albumen, the cross protection between serotype can be triggered.Currently available vaccines, existing vaccines method almost only relies only on the immune of O antigens mediation, Using five kinds or six kinds of serotypes provide for most places and epidemic dysentery case it is highly protective the fact.But It is, it is contemplated that most of polyvaccines suggested are based on purifying subunit (O antigens) or several have different LPS O antigenic types Attenuated live bacterium the fact, their (being likely to) are too complicated and therefore expensive.It is based on further, since being produced to vaccine serotype The popular increase of the immune pressure that drove is immunized, escape and nonvaccine serotype, the covering expection of part serotype cause serum Type substitutes, as indicated in other more serotype cause of disease such as streptococcus pneumonias.Therefore, preferable shigella vaccine is expected to carry For a large amount of cross protections for all circulation serotypes.

In addition to Shigella, enterotoxic Escherichia coli (ETEC) is the main bacteria pathogen of passenger's diarrhea, and It is one of underlying cause of death of endemic disease country death of child.Therefore, people take effort to develop while solve both pathogen Vaccine.

The diarrhea of passenger passes through antibiosis extract for treating at present；But the resistance of Shigella bacterial strain is risen so that the disease Processing it is more and more difficult.In addition, ETEC the infected can by with the relevant long-term consequence of intestinal irritable syndrome.Connect extensively Receive, vaccine inoculation is to solve the most economical approach of this highly unsatisfied medical demand；But currently without prevent this The vaccine of a little situations.

Two kinds of enterotoxins, thermal instability toxin (LT) and heat endurance toxin (ST) have been identified in ETEC bacterial strains, has been made For with the relevant ST of pig disease (STp) or with the relevant ST of human disease (STa).LT in structure with cholera toxin very high homology.A Subunit is the active component of the toxin, it is acted on to increase the activity of adenyl cyclase.This is delivered to host by B subunits Into the cell, B subunits are attached to the gangliosides on cell surface.STa is that small (19 amino acid) non-immunogenic is more Peptide, it is with guanylate cyclase Activation Activity.STm is the mutant form of ST nontoxic but still with immunogenicity.It is this STm is considered that vaccine antigen (Taxt et al.Infect.Immun.78 can be employed as safely:1824-31,2010).

It was demonstrated that ETEC bacterial strains also produce EAST1, it is a kind of similar with ST in size and binding mode but in sequence not Same heat endurance toxin, initially identifies (Nataro and Kaper, Clin in enteroaggrerative E.coli bacterial strain Microbiol Rev.11:142-201,1998；Zhang et al.,Vet Microbiol.123：145-152,2007).

Zheng et al. (World J.Gastroeneterol.11：3411-18,2005) construct asd mutant will he Salmonella bacterial strain, it co-expresses the CS3 and LTB/STm of enterotoxic Escherichia coli.After mouse is immunized by peroral route, there is pin To the antibody of CS3, LTB, ST and Shigella lipopolysaccharides.

Xu et al. (Vaccine 21:664-648,2003) describe attenuation invasion Shigella flexneri serotype 2a living Carrier of the rfbF mutant as the HIV gag vaccines based on DNA.

Noriega et al. (Infection and Immunity 67 (2):782-788,1999) describe for 14 kinds The cross protection strategy of Shigella flexneri serotype, includes the use of two kinds of serotypes 2a and 3a.The attenuated strain of description is Fu Shi Shigella dysenteriae 2a bacterial strains CVD1207 (Δ guaB-A Δ set1 Δ sen) and 1211 (Δ guaB-A Δs of Shigella flexneri 3a bacterial strains CVD virGΔsen)。

Bernardini et al. (Infection and Immunity 69 (2):1072-1083,2001) describe Fu Shi The mutant of shigella dysenteriae 5, it is aroC mutant and double purE aroC mutant.

Levine et al. (Behring Institute Mitteilungen 98:120-123,1997) describe Fu Shi Shigella dysenteriae 2a attenuated strains CVD 1203, and Shigella flexneri 2a candidate vaccines CVD 1205, CVD 1203 includes chromosome The mutation of gene aroA and plasmid gene virG, the deletion mutation that CVD 1205 includes guaB-A (make it in terms of nucleic acid synthesis It is defective) and virG deletion mutation.

It is an object of the present invention to provide improved shigella vaccine, is particularly used for the trip for preventing and going endemic disease country The diarrhea disease of child's height correlation that is objective and staying in developing country.These vaccines can be based on heterologously expressed and be obtained from The antigen of different pathogens simultaneously causes the attenuation Fu Shi will protected extensively of directed toward bacteria pathogen especially Shigella to be congratulated Bacterium live vaccine strain.

The content of the invention

This target can be realized by claimed theme.

The present invention provides Shigella attenuated live vaccine, it is based on the coarse Shigella bacterial strain for lacking LPS O antigens.

Particularly, it is Non-Invasive that Shigella bacterial strain is particularly attacked after plasmid encoding mutant by mutation.

Especially, the mutation for one or more genes that vaccine of the invention includes in being synthesized, transported and being expressed by LPS And be attenuated, the gene is preferably selected from the gene in rfb manipulation submanifolds, and the rfb operators are for example positioned at Shigella flexneri 2a The Gnd (6-phosphogluconate dehydrogenase, 2089155-2090561) and GalF (UTP- Cori esters of 2457T bacterial strains Uridyltransferase, 2101928-2102821) (Wei et al.Complete genome on chromosome between gene sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T.Infect Immun.2003May；71(5):2775-86.) or on pathogenic plasmid (sonnei shigella dysenteriae).Especially Preferably following mutation：One or more genes in the rfb/wbb gene clusters of O antigens synthesis are encoded, coding O antigens connect The waaL of enzyme is met, encodes the wzx of the O antigen flippases included in O antigen deliveries, the wzy/rfc included in the polymerization of O antigens, is compiled Gene in the rfa/waa gene clusters of code LPS core synthesis, influences the adjusting gene of O antigen presentations, such as rfaH, or missing O antigen presentations are caused to reduce by least 90% function.

According to a specific embodiment, the mutation is one or more deleted in rfb F, D, C, E, J and/or I It is a, or one part is deleted, or delete corresponding gene in different Shigella serotypes.Alternatively, it can use by passivation Mutation, such as temporarily passivation, conditionally passivation or composition passivation.

The Shigella bacterial strain is preferably selected from Shigella, is selected from any Shigella serotype or kind, special It is not Shigella flexneri, sonnei shigella dysenteriae, Shigella dysenteriae and shigella boydii.

Especially, the Shigella bacterial strain expression can cause the outer membrane protein of cross reacting antibody, particularly conservative Albumen, including OmpC, OmpA and OmpX, or those albumen of coding on invasion and attack plasmid.

The vaccine of the present invention is to different serotypes and Shigella not of the same race, particularly to Shigella flexneri 2a, Fu Shi The one of any of shigella dysenteriae 6 and sonnei shigella dysenteriae or enteroinvasive E.Coli especially has intersecting protective.

According to a specific aspect, the Shigella is mutated by attacking the further of plasmid, is particularly deleted The mutation of the invasion and attack plasmid of ipaB and/or ipaC and/or other ipa genes, and become Non-Invasive.

Especially, the Shigella includes restructuring endogenous invasion and attack plasmid, and the plasmid includes encoding heterologous antigens At least one gene, to secrete the antigen or by the antigen presentation in bacterial cell surface.

Preferred embodiment is related to this vaccine, wherein the antigen is the protective antigens obtained from pathogen, the disease Substance is selected from：

- bacterial antigen, preferably toxin or colonizing factor,

- viral antigen, is preferred from causing enteral or the cause of disease of mucosal infections,

- fungal allergen, is preferred from causing enteral or the cause of disease of mucosal infections, and

- parasitics antigen, is preferred from the cause of disease for causing intestinal infection.

Especially, bacterial antigen is derived from enteropathic bacteria, is preferably selected from：

A. bacillus coli antigen, is especially selected from following enterotoxin：The LTB of ETEC, the LTA and ST, subunit of mutation, Or its fusion, the antigen from enteroinvasive E.Coli (EAEC), or shiga-like toxin 1 or 2

B. campylobacter jejuni antigen,

C. clostridium difficile antigen, particularly toxin A and B

D. comma bacillus antigen, particularly CT-B antigens, and

E.a), mutant or fusion protein b), c) or d).

Especially, bacterial antigen is an enterotoxin (ETEC), and it includes heat-labile toxin B subunits (LTB), heat are steady Determine toxin (ST) or subunit or their fusion, preferably include the STm with the amino acid sequence as shown in SEQ ID 1 LTB/STm, it does not include the wild-type sequence of people ST alternatively.

Particularly, the ETEC antigens are the unit B of LT and the fusion protein of mutant ST, preferably a fusion protein LTB/ STm, its amino acid sequence are obtained from Fig. 7 SEQ ID 11-18 (LTA- promoter-LTB-ST-LTB terminator nucleosides of 4 constructions Acid and LTB-ST amino acid sequences), such as the ST mutant at position 13 and/or 12, such as P13F or P13G, and/or N12R or N12K).

Especially, viral antigen comes from diarrhea virus, is preferably selected from rotavirus and norwalk virus (calicivirus).

Especially, parasitics antigen comes from the protozoan for causing diarrhea, is preferably selected from giardia lamblia stiles, Cryptosporidium Kind and Entamoeba histolytica.

Especially, fungal allergen comes from the fungi for causing diarrhea, is preferably selected from Blastomyces dermatitidis and Histoplasma.

A specific aspect according to the present invention, shigella vaccine bacterial strain further comprise deleting for necessary chromogene Remove, and the gene is inserted into invasion and attack plasmid, particularly ppa genes, or accD, acpS, dapE, era, frr, ftsI, FtsL, ftsN, ftsZ, infA, lgt, lpxC, msb, murA, murI, nadE, parC, proS, pyrB, rpsB, trmA, rho With any gene in rhoL.

However, a specific embodiment according to the present invention, which provides the prevention or immunoprophylaxis for main body To prevent infectious diseases, particularly intestines problem, such as diarrhea disease.Especially, the disease is selected from shigellosis, dysentery And diarrhea.

Present invention also offers the method for infectious diseases particularly intestines problem in prevention main body, especially by institute Main body is stated to carry out vaccine inoculation respectively and be immunized to prevent.

Especially, the intestines problem is caused by any Shigella serotype or kind.

Preferably, described (immune) prevention includes giving vaccine with mucous membrane or oral formulations.

Especially, the vaccine it is oral or nasal in give.

One specific embodiment is related to vaccine used according to the invention, wherein

The protectiveness that-polyvaccine is used for expressing one of Shigella and at least one kind in addition to Shigella resists Original, it is to be attacked by the way that the protective antigens of the pathogen to be brought into the restructuring through endogenous sex modification in plasmid, Yi Jiqi In

- the infectious diseases is caused by any Shigella serotype or kind and/or the pathogen.

Another aspect provides Shigella bacterial strain, it is Shigella flexneri 2a bacterial strains, such as Fu Shi will is congratulated Bacterium 2a2457T, it deletes rfbF, and at least one of ipaB and/or ipaC genes, or deletes its necessary part.

Especially, the Shigella bacterial strain can further comprise the deletion and gene insertion of necessary chromogene Into invasion and attack plasmid.

Preferably, the Shigella bacterial strain includes a restructuring invasion and attack plasmid, and the plasmid includes encoding heterologous antigens At least one gene to express and/or secrete the antigen.

However, the recombinant plasmid that the present invention further aspect provides the Shigella invasion and attack plasmid based on mutation carries Body, the invasion and attack plasmid include the nucleotide sequence for encoding at least one heterogenetic antigen, wherein the plasmid in ipaB and/or At least one ipaC genes place is mutated.This especially refers to that to delete and/or be passivated non-coding or coding region respectively (such as operable Ground is connected to the regulating and controlling sequence and a gene of a gene) mutation, preferably assign the base of bacillary host cell Non-Invasive Because deleting, ipaB and/or ipaC genes are particularly deleted, or delete its (necessity) part.

Further specific aspect of the invention is related to the bacillary host cell for including carrier of the present invention, wherein the host Cell is in particular selected from Shigella, Escherichia coli, salmonella, campylobacter or Yersinia.

The host cell particularly comprises the mutation of endogenous invasion and attack plasmid.Especially, carrier of the invention is endogenous Property invasion and attack plasmid, i.e., be endogenous or the plasmid of homology for host cell.

Brief description of the drawings

Fig. 1Lack the Shigella flexneri of LPS O antigens synthesis and five kinds of attenuation Non-Invasive viable bacterias of sonnei shigella dysenteriae The cross-protection ability of strain.

With the CRP (10 of Shigella flexneri 2a (a and b) mutant⁶) and CRN (10 CFU⁸CFU) variation or sonnei will are congratulated The I phases (10 of bacterium (c)^5.5) and the II phases (10 CFU^7.5CFU) variation by the group of 8 week old BALB/c mouse of intranasal immunisations twice, in Between be spaced two weeks.Control group salt water modeling vaccine inoculation.Then, with 10⁶The wild type Shigella flexneri 6 (a and c) of CFU or 10^6.5The wild type sonnei shigella dysenteriae (b) of CFU attacks mouse by identical approach.Then detection survival rate 14 days.Attached drawing is shown The merging data of three (b) or two (a and c) independent experiments, every group of 5 mouse are shown, and have repeated to test.Use Log-rank (Mantel-Cox) examine and statistical analysis is carried out to Survival curves.The mouse of simulation vaccine inoculation is markedly different from survival rate In the case of, p value is shown on the diagram.

Fig. 2 institutesThe difference of antibody level caused by the antigenic phenotype of the different mutants used and their possibility Schematic diagram.

The antibody of O antigens, its gene determinant and their triggerings is shown in figure.Ipa and secondary guarantor are shown in figure The surface antigen kept.Express mutant (Shigella flexneri Δ aroC CRP and the sonnei shigella dysenteriae I of both Ipa and O antigens Phase) triggering mainly for these major antigens antibody response.In contrast, these antigens are lost in vaccine strains can cause pair The higher reaction of secondary conservative antigen.ip：Attack plasmid, chr.：Chromosome, T3SS：Three type excretory systems, LPS：Lipopolysaccharides.

Fig. 3Wide spectrum reactivity mucous membrane IgA obtained from the mouse with Shigella flexneri 2a Attenuated strain vaccine inoculations.With In the BAL samples collected after smooth ipa positive strains (Δ aroC CRP) and double mutant (Δ rfbF CRN) are 2 times immune The immunoreactivity of sIgA is determined with ELISA on different target bacterial cell.Reactivity is expressed as the ratio under identical dilution factor Rate (reactivity of Δ aroC CRP samples divided by the reactivity of Δ rfbF CRN samples) is so as to repeat comparable.Attached drawing is shown The standard deviation of average+average of four experiments carried out with the BAL samples obtained from independent vaccine inoculation.Data pass through Mann- Whitney non-parametric tests are to dual (ipa and O antigens) Positive Objects (Δ aroC CRP；Black column) on the value that obtains carry out Statistical Comparison.

Fig. 4Supplementary table 1：For producing and determining the oligonucleotides of deletion mutant.(SEQ ID 3–10)

Fig. 5It is attenuated the schematic diagram of Shigella flexneri 2a 2457T bacterial strains.

A. bacterial strain is sequenced in Shigella flexneri 2a 2457T entirely.O antigens are the serotype determinants of Shigella.Shigella The essential factor of synthesis of rfbF gene codes on chromosome to the O antigen components of LPS.All pathogenicity Shigellas The plasmid-encoded invasion and attack plasmid antigen (IpaA-D) of invasion and attack present in bacterial strain, it is important point of type III excretory system (T3SS) Subgroup point.The critical process of T3SS mediations target cell-Shigella interaction, and ultimately result in the transfer of the Shigella factor Into target cell, pathogen is set to attack and spread.Ipa B and IpaC are the necessary components of key of this system.

B. in Shigella flexneri 2a T2457rfbF^-ipaB/C^-Introduce in mutant and deleted at two.Deleted from chromosome The synthesis of O antigens except rfbF gene removals, and " coarse " bacterial strain for being attenuated, and cause serotype only in vaccine inoculation Vertical is immune.The deletion of ipaB and C genes destroys T3SS on invasion and attack plasmid, so as to assign Shigella mutant Non-Invasive (and Congo red feminine gender).

C. in order to stablize the invasion and attack plasmid of mutation Shigella bacterial strain, necessary chromogene is deleted from chromosome (inorganic pyrophosphatase, ppa (Fig. 7 SEQ ID 24)), and it is reintroduced to invasion and attack plasmid as a part for synthesis tectosome In.

Fig. 6：With ipa clusters, for the ipaB/C primer locations (pKD1,2) deleted and for monitoring ipaB/C deletions Compare the schematic diagram of the invasion and attack plasmid pCP301 of 301 bacterial strains of Shigella flexneri 2a of primer (ko1,2).Also designate ipaJ and The position of the insertion point of synthetic gene between IS100 elements.

Fig. 7：Sequence

The gene of LT-B/mST fusion proteins, its encode have from amino acid position 13 (from Pro to Phe) ST of mutation with And eltAB promoters and terminator sequence

GP-P13F (nucleotide sequence, SEQ ID 11),

LT-B/mST fusion proteins, have mutation in ST at amino acid position 13 (from Pro to Phe)

GP-P13F (amino acid sequence, SEQ ID 12),

The gene of LT-B/mST fusion proteins, its encode have at amino acid position 13 (from Pro to Gly) ST of mutation with And eltAB promoters and terminator sequence

GS-P13G (nucleotide sequence, SEQ ID 13),

LT-B/mST fusion proteins, have mutation at the amino acid position 13 of ST (from Pro to Gly)

GS-P13G (amino acid sequence, SEQ ID 14),

The gene of LT-B/mST fusion proteins, its encode have at amino acid position 12 (from Asn to Arg) ST of mutation with And eltAB promoters and terminator sequence

GS-N12R (nucleotide sequence, SEQ ID 15),

LT-B/mST fusion proteins, it has mutation at the amino acid position 12 of ST (from Asn to Arg)

GS-N12R (amino acid sequence, SEQ ID 16),

The gene of LT-B/mST fusion proteins, its encode have at amino acid position 12 (from Asn to Lys) ST of mutation with And eltAB promoters and terminator sequence

GS-N12K (nucleotide sequence, SEQ ID 17),

LT-B/mST fusion proteins, its ST have mutation at amino acid position 12 (from Asn to Lys)

GS-N12K (amino acid sequence, SEQ ID 18),

For confirming that ipa deletes the positive control PCR primer of mutant strain

Ipa co1 (SEQ ID 19),

For confirming that ipa deletes the reverse control PCR primer ipa co2 (SEQ ID 20) of mutant strain,

To produce the positive PCR primer that ipa deletes mutant strain

Ipa pKD1 (SEQ ID 21),

To produce the positive PCR primer that ipa deletes mutant strain

Ipa pKD2 (SEQ ID 22),

The nucleotide sequence of the ipaB and ipC genes removed from invasion and attack plasmid

ipaBC(SEQ ID 23)。

The nucleotide sequence of the ppa genes of invasion and attack plasmid is transplanted to from chromosome

Shigella ppa genes (SEQ ID 24).

To produce the positive PCR primer that ppa deletes mutant strain

ppa pKD-F(SEQ ID 25)

To produce the inverse PCR primer that ppa deletes mutant strain

ppa pKD-R(SEQ ID 26)

For confirming that ppa deletes the positive control PCR primer of mutant strain

ppa ko1(SEQ ID 27)

For confirming that ppa deletes the reverse control PCR primer of mutant strain

ppa ko2(SEQ ID 28)

Be inserted between LT-B and mST for the connection peptide that flexibly folds

GGGGS(SEQ ID 29)

Fig. 8：

Delete the PCR amplification of the chromosomal region of rfbF genes.M：DNA size labels；WT：Wild type Fu Shi will is congratulated Bacterium 2a 2457T, the rfbF genes with flank region, 1100bp fragments；mt：Δ rfbF mutant, is substituted with chloromycetin gene Gene, 1300bP.

Fig. 9：

Delete the PCR amplification in the invasion and attack plasmid region of ipaC and ipaB genes.M：DNA size labels；WT：Wild type Shigella flexneri 2a 2457T, ipaB the and ipaC genes with flank region, 1600bp fragments；mt：Δ ipaBC mutation invasion and attack Plasmid, the gene substituted with kanamycin gene, 2570bp.

Figure 10：

A. the knot of the polygenes tectosome of essential gene Ppa, LTB-mST fusion protein and kalamycin resistance albumen is encoded Structure.The expression of LTB-mST fusion proteins is driven by LTA promoters, and transcription is terminated with LTB terminators.4 with removing toxic substances ST mutation The LTB-ST amino acid sequences (P13F, P13G, N12R, N12K) of a tectosome are marked that (codon of mutation has added lower stroke Line).Abbreviation：CS：Cloning site；H1 and H2：Homology region 1 and 2 on invasion and attack plasmid is to help homologous recombination；ppa：It is inorganic Pyrophosphatase gene；pro：Promoter；term：Terminator；GGGGS：Gly-Gly-Gly-Gly-Ser (SEQ ID 29LTB and Pentaamino acid connexon between mST；kan：Kalamycin resistance gene.

B.ETEC genes are inserted into Shigella invasion and attack plasmid.M：DNA size labels；WT：Wild type Fu Shi will is congratulated Bacterium 2a 2457T, attack plasmid gene interval region, 450bp fragments；mt：LT-B+STm gene mutations attack plasmid, with card that The gene of mycin gene substitution, 2800bp,

C. immunoblotting assay is to detect LTB.Restructuring LT-B, and Escherichia coli and Shigella are separated with SDS-PAGE Culture supernatant part (albumen of secretion), will detect on protein delivery to nitrocellulose filter and with anti-LTB monoclonal antibodies.Swimming Road 1：Wild type Shigella flexneri (negative control)；Swimming lane 2：Attack in plasmid and carry the Shigella flexneri of fusion；Swimming lane 3：The DH5 α Escherichia coli converted with pGET carriers, it includes synthesis construction as shown in Figure 10 A；Swimming lane 4：Express the ETEC of LT Bacterial strain (positive control).

Figure 11：

The heterologous as caused by Shigella flexneri 2a vaccine strains is protected, and the bacterial strain is common through rfbF and ipaC/ipaB The transformation of mutation.The each group of 5 mouse wild-type strain Shigella flexneri 2a 2457T (5 × 10⁵Cfu/ mouse) or its etc. (all be 10 to gene elmination mutant 2457T Δs rfb, 2457T Δ ipaBC, 2457T Δ rfb Δs ipaBC⁸Cfu/ mouse) Sublethal dose intranasal be immunized, or with PBS buffer to carry out simulation immune.Carry out identical immune three times, interval is 2 Week.Last time booster immunization latter week, with the sonnei shigella dysenteriae bacterial strain (2 × 10 of lethal dose⁶Cfu/ mouse) attack it is small Mouse.The survival rate of daily monitoring animal.

Embodiment

Proprietary term has following meanings throughout the specification.

Terms used herein " attenuation " describes the pathogenic strain of modified Shigella so that it is no longer able to draw Play disease, i.e. modified bacterial strain is nontoxic.It is used herein as with the relevant term of attenuated shigella " work " describe can Growth and the Shigella of breeding.Therefore, work Shigella bacterial strain of the invention is used in attenuated live vaccine and is particularly capable of Be colonized, but do not cause and the relevant clinical symptoms of intestines problem in the large intestine of main body (enteron aisle or diarrhoea pathogenic can cause).This Outside, live strain of the invention is particularly capable of limitedly replicating in the main body for be vaccinated with vaccine, and can cause prevents will from congratulating The protective immunological reaction of the pathogenic strain of Salmonella.The attenuated bacteria of the present invention can will not to express natural bacteria through genetic modification The heterogenetic antigen of expression so that carrier of the attenuated bacteria as heterogenetic antigen.

Terminology used in the present invention " antigen " should refer in particular to any antigenic determinant, it is possible to by the binding site of antibody Identification.Particularly preferred antigen is to have confirmed to be or can become those immune or treatment-related molecules or structure, especially It is those of clinical efficacy after tested.Term as employed herein particularly including the molecule or structure selected from antigen, it is described anti- Original is comprising immune accessible and immune associated epitope, the conservative antigen particularly found in one or more kinds or serotype. Immune accessible epitope is usually presented or is contained therein by the antigen expressed on cell surface.Term " protection used herein Property antigen " should refer to triggering vivo immunization reaction with cause be directed to antigen neutralizing antibody those antigens.This is provided with anti- Original carries out effective protection during active immne.Proteantigen is preferable antigen, causes cell and body fluid to be exempted from because they have The capability of epidemic disease reaction.

Term " heterologous " here especially uses antigen, and is interpreted as carrying out the cell for expressing the heterogenetic antigen Say be exotic antigen.For Shigella cell or bacterial strain, which especially refers to for Shigella cell or bacterial strain For be respectively exotic antigen.Therefore, this heterologous or exotic antigen will not express in wild-type cell or bacterial strain, But recombinant includes the heterologous gene for encoding the antigen.So as to which the recombinant cell or bacterial strain can resist the heterologous Original expression is for example on cell surface.The cell of the present invention can be through genetic modification with expressing heterologous antigen, such as LT and/or ST Or the nontoxic component or form of STm.These cells cause the immune response for heterogenetic antigen and native antigen, and therefore The protection provided by vaccine is provided.

Term " cross reaction " used herein on antigen should refer to the anti-of the epitope shared between Different Kinds of Pathogens Original, includes for example mutually of the same race or different bacterium kind different serotypes.Cross reacting antigen is typically conserved structure.Intersect anti- Epitope is answered to may originate from the same antigen of Different Kinds of Pathogens expression, or from the not synantigen with similar structures.

Specific cross reacting antigen identified by cross reacting antibody, such as sero-fast antibody, separated antibody or again The antibody of group.These cross reacting antibodies can recognize that the cross reacting antigen of different pathogens.Specific cross reagin is Neutralizing antibody.

" cross protection " vaccine or immune response be interpreted as by least one different pathogen it is for example not of the same race or Serotype (its with cause reaction used in differ) come prevent infection vaccine or immune response.Cross protection effect is usual It can be tested with the different antiserums from the main body exposed to different pathogens.

When cross reacting antigen design is to cause cross protection to be immunized, this can be tested in animal model, such as with obtaining Animal triggering immune response is immunized from a kind of cross reacting antigen of pathogen, and causes the disease of the reaction with being different from At least one pathogen of substance attacks animal.As an example, it is this to be directed to more than one Shigella serotype or other The cross protection of intestines invasion and attack bacterium such as Escherichia coli with cross protection antigen, which is immunized especially to refer to, prevents Different Individual Different variants in the kind of bacterium, such as the variation of sub-species.The group of serovar with common antigen is known as sero-group Or serotype.

It is generally to deposit to develop for example more bacterial strains of wide spectrum and/or the basis of more serotypes and/or multiple types shigella vaccine In the identification of the cross reacting antigen in Shigella serovar.This especially includes infecting relevant separation strains with people.

When polyvaccine design is to cause the cross protection for Different Kinds of Pathogens to be immunized, immune response is usually by several anti- Original causes, such as the various antigens from Different Kinds of Pathogens.As an example, this polyvaccine can based on from it is not of the same race at least Two kinds of protection antigens, such as obtained from Shigella and Escherichia coli, it is also possible to obtained from other bacteriums, virus, fungi or parasitism Property antigen.The polyvaccine of cross protection can for example be tested in animal model, by using comprising obtained from least two different diseases The different protection antigen-immunized animals triggering immune responses of substance, and with one kind, two or more following pathogen challenge animals.

The basis of multiple types candidate vaccine of the exploitation based on specific attenuated shigella bacterium (as used in the present invention) Identification protective antigens, its either cross reactivity to tackle a series of different serotypes, or be not intersect it is anti- Answering property, its generally existing in the kind of Different Kinds of Pathogens, is to seek protection targeted object.

As employed herein be especially also referred to as " in enteron aisle ", Ying Zhiyu with disease or the relevant term of pathogen " enteral " Enteron aisle in relation to or influence the disease condition or pathogen of enteron aisle, such as dysentery or diarrhea disease.Especially, this intestinal bowel disease refers to The infectious disease of colon.Specific symptom is including bleeding, the diarrhea full of mucus；Abdominal pain；Fever or loss body fluid.Diarrhea disease Refer to cause three times a day or repeatedly loose stool or liquid manure, or defecation to be more than the situation of the normal number of the people.Enteropathogen It is interpreted as causing main body to suffer from intestines problem when main body is poisoned when with the pathogenic infection or with toxin particularly enterotoxin.

Enteric infection disease such as dysentery or diarrhea have many causes, including virus, bacterium, fungi and parasite.Example There is provided as follows：Norovirus is adult's most common cause of virus diarrhea, but rotavirus is less than five years old children virus The most common cause of diarrhea.40 and 41 type adenovirus and astrovirus cause substantial amounts of infection.Bacterium campylobacter is bacterium The common causal origin of property dysentery or diarrhea, but the bacterium of salmonella, Shigella and some escherichia coli (Escherichia coli) Infection caused by strain frequently occurs on some areas.In the elderly, particularly those use the uncorrelated infection of antibiosis extract for treating In the elderly, the toxin that clostridium difficile produces often causes severe diarrhea.The example of parasite includes that the merchant of chronic infection can be caused Flagellate, and Entamoeba histolytica.The soluble exemplary intestines problem of vaccine of the present invention be shigellosis and The relevant diarrhea of ETEC.

The term " endogenous " used herein in relation to plasmid should refer to the plasmid from particular host cell.Endogenous plasmid Restructuring endogenous plasmid can be obtained through genetic modification, such as by recombinant technique with original position transformation plasmid, i.e., comprising natural In the host cell of endogenous plasmid, or from host cell remove plasmid when, it is received laboratory operation, be then reintroduced back to In the host cell of same type.The invasion and attack phenotype of Shigella is especially assigned by the endogenous 220-kb plasmids that cause a disease, should Plasmid also referred to as attacks plasmid, or natural or endogenous invasion and attack plasmid.Shigella endogenous invasion and attack plasmid particular according to The present invention is provided to recombinate purpose, as separated invasion and attack plasmid or for in-situ restructuring.

The term " necessity " used herein in relation to gene is interpreted as referring to biological existence (such as bacterial cell duplication) living Required gene.The mutation of essential gene, such as delete and/or be passivated, lethal phenotype or not replicated cell can be caused.Shiga The essential gene of bacterium may be mutated to delete the gene of Shigella chromosome, and further bring gene in invasion and attack plasmid into Plasmid is attacked to stablize.This provides the culture with the Shigella for stablizing restructuring endogenous invasion and attack plasmid.Shigella Essential gene has ppa, accD, acpS, dapE, era, frr, ftsI, ftsL, ftsN, ftsZ, infA, lgt, lpxC, msbA, MurA, murI, nadE, parC, proS, pyrB, rpsB, trmA, rho and rhoL gene.

Herein, " passivation " of gene is always understood to refer to the temporary transient of gene, induction or composition downward, to reduce or suppress The expression of gene outcome.This especially by gene or can be operably connected to the regulatory sequence of the gene and for example adjust gene The mutation of the promoter of expression, enhancer etc. is completed.In passivation mutation, particularly existing those causes polynucleotides or gene Such as the mutation that the expression of the gene of coding virulence factor is reduced or suppressed, or cause each non-functional albumen such as non-functional The mutation of virulence factor expression.

The term " invasion and attack " or " non-invasion and attack " used herein in relation to gene understands in the following way.Attack pathogenetic bacteria Eukaryotic can be attacked.For example, after invasion and attack, Shigella can in the cell multiply and be diffused into neighbouring epithelial cell, lead Cause the pathological characters of disorganization and shigellosis.In the gene for adjusting Shigella invasiveness, there is such as coding and invade Attack the ipa genes of plasmid antigen.The deletion and/or passivation of these at least one genes can cause non-invasion and attack Shigella.

Sereny experiments are to determine the code test (Wood of the invasiveness of organism such as Shigella or Escherichia coli et al.J.Clin.Microbiol.24：498-500,1986).This is completed by the way that bacterial suspension is inoculated into guinea pig eye 's.Serious coctum and serious keratitis show positive test.

The art used herein of invasion and attack plasmid on nucleic acid and in particular, to carrier, plasmid particularly Shigella This compound that language " separated " or " separation " Ying Zhicong naturally are sufficiently separated out in relevant environment, so that it is with " base This is pure " form exist.Term " substantially pure " used herein or " purifying ", which should refer to, includes at least 50% (w/w), preferably at least 60%th, the prepared product of 70%, 80%, 90% or 95% compound such as nucleic acid molecules or plasmid.Purity is by being suitable for this The method (such as chromatography, polyacrylamide gel electrophoresis, HPLC analysis etc.) of compound measures.

" separated " need not refer to exclusion manually or synthetic mixture, the mixture have other compounds or material, or In the presence of do not disturb primary activity and for example due to not exclusively purifying and existing impurity.Especially, the separated nucleic acid of the present invention Molecule also refers to including those chemical syntheses.

When mentioning separated invasion and attack plasmid of the invention, this term refers to the separated plasmid from the cytoplasm to be originated from." point From plasmid " can further indicate that by biology or the plasmid that directly produces of synthetic method and during its generation it is existing Separated plasmid in other components.

The term " mutation " used herein in relation to gene should refer to the mutation of gene order, in particular at least one nucleotide Any deletion, substitution, insertion, or any combination thereof, to obtain mutator.This should refer in particular to whole gene or its big portion Point, such as delete at least 50% gene.Term " mutation (mutagenesis) " and " mutation (mutation) " are exchanged here Use.

Refer to the relevant term of gramnegative bacterium such as Shigella " coarse " rough, and should especially include " slight coarse " (i.e. O-ag synthesis is lowered) or " deep rough " bacterium.Term " coarse " used herein may include feature for example Irregular colonial morphology simultaneously may include such as waveform and/or leaf-like morphology.The term, which especially refers to, cannot and/or substantially cannot Produce the bacterial strain of O polysaccharide.The repetition polysaccharide polymer included in LPS is referred to as the O antigens, O polysaccharide or O (side) chain of bacterium.O resists Original is connected to core oligosaccharide, and the outermost layer domain comprising LPS molecules.O chains, which are deposited to be not present, determines that LPS to be considered as coarse or light It is sliding.For the bacillary bacterial strain that O antigenic structures have changed when being grown on agar plate, their appearance is changed into dull from smooth.Entirely Long O chains make LPS smooth, and O chains lack or shortening makes LPS coarse." smooth " bacterium includes complete core and O antigens.It is " thick It is rough " bacterium includes the shortages of LPS O antigens, it is meant that shorten without the chain length of O antigens or O antigens or the number of smooth LPS chains Mesh is reduced.Term " slight coarse " refers to the subgroup of coarse bacterium, its O antigens or quantity with chain length reduction reduces smooth LPS chains." deep rough " bacterium loses part LPS cores, therefore also lacks O antigens.

Term " lacking LPS O antigens " used herein on coarse Shigella should especially refer to as code test is true Fixed, less than 50%, or less than 40%, or less than 30%, or less than 20%, or less than 10%, or resist substantially without or without LPS O It is former.

Code test can be used to determine the rough features of bacterial strain.

For example, the phenotype of LPS mutant can be for example by the SDS-PAGE separation of LPS and silver staining or special using serotype The agglutination test of different immune serum determines.

Coarse Shigella can be produced by being attenuated, for example, by least one gene or its most mutation, such as By deleting and/or being passivated, the gene is included in LPS synthesis, transhipment and/or expression, is preferably selected from rfb and is manipulated in submanifold Gene, or one or more genes in the rfb/wbb gene clusters of coding O antigens synthesis, coding O antigen ligases WaaL, coding are included in the wzx of the O antigen flippases in O antigen deliveries, the wzy/rfc included in the synthesis of O antigens, coding Gene in the rfa/waa gene clusters of LPS cores synthesis, influences the adjusting gene of O antigen presentations, such as rfaH, or loses and lead O antigen presentations are caused to reduce by least 90% function.

The specific gene example included in LPS sugar synthesis is rfbA, B, D and C.

The specific gene example included in LPS sugar transferases is rfbF and G.

The specific gene example included in LPS O antigen polymerases is rfc/wzy.

Rfb manipulates submanifold and is located on chromosome or on invasion and attack plasmid (sonnei shigella dysenteriae).Specific base in this cluster Because being rfb F, D, C, E, J and/or I gene.

As used herein, term " restructuring " refers to the natural molecule or construction being not present in host cell.In some realities Apply in scheme, recombinant nucleic acid molecules include two or more naturally occurring sequences, it is connected in a manner of non-naturally occurring Together.Recombinant protein refers to the albumen for being encoded by recombinant nucleic acid and/or being expressed.In some embodiments, " recombinant cell " is expressed In the intracellular gene that can not find same form of natural (that is, non-recombinant) form, and/or expression can generally be overexpressed extremely, Low expression and/or not natural gene of the expression (due to intentional human intervention).Recombinant cell includes at least one restructuring Polynucleotides or polypeptide." restructuring ", " in restructuring " and generation " restructuring " nucleic acid generally include at least two nucleic acid fragments of assembling. In certain embodiments, recombinant protein and recombinant nucleic acid are still functional, i.e. retain their work in host cell Property or display enhancing activity.Under any circumstance, it is thin to be considered as restructuring for attenuated bacteria attenuated shigella of the invention Born of the same parents.Nucleic acid construct such as plasmid or carrier, nucleic acid (such as polynucleotides), polypeptide or host cell herein referred to as " recombinate ", When it is not naturally occurring, when being artificial or engineered.Shigella restructuring invasion and attack plasmid especially it is engineered with comprising One, the specific deletion and/or passivation of two or more polynucleotides or gene, for example, at least one coding invasion and attack plasmid antigen Gene deletion, and/or further include one or more heterologous genes, such as the gene of encoding protective antigens.

" stabilization " restructuring invasion and attack plasmid of Shigella is such a Shigella plasmid, it is being selected to remain thin Show in Shigella cell culture at least 50% under conditions of plasmid in born of the same parents' culture, at least 60%, at least 70%, At least 80%, at least 90% or the reservation more than 90%.Shigella stablize restructuring invasion and attack plasmid a particular instance be Mutated deletion and/or passivation are located at the essential gene of chromosomal loci, and the restructuring integrated at invasion and attack plasmid gene seat Shigella host cell.Shigella without invasion and attack plasmid will not grow or will not replicate, and attack plasmid comprising endogenous Shigella can grow and replicate in vivo.

As used herein, term " carrier " refers to an instrument, and DNA or for example external (heterologous) gene of RNA sequence can be borrowed To introduce host cell, to change cell and promote the expression (such as transcription and translation) of introduced sequence.Plasmid is the present invention The invasion and attack plasmid of preferable carrier, particularly Shigella, especially attacks plasmid including endogenous.

Carrier generally comprises the DNA of heritable medium, and foreign DNA can be inserted into wherein.One DNA fragmentation is inserted into another A kind of common method in DNA fragmentation includes the use of the enzyme of referred to as restriction enzyme, certain bits of the restriction enzyme in referred to as restriction site Point (specific nucleotide group) crack DNA." box " refers to the DNA of coded sequence or coding can be inserted into the expression of the restriction site limited The DNA fragmentation of product.The purpose of the restriction site design of box is to ensure that box gene is inserted into appropriate reading frame.Generally For, foreign DNA is inserted at one or more restriction sites of carrier DNA, is then carried by carrier with heritable carrier DNA enters host cell.DNA fragmentation or sequence with insertion or increased DNA, also referred to as such as expression vector, " DNA Tectosome ".A kind of common type of carrier is " plasmid ", it is typically the self-contained molecule of double-stranded DNA, is usually bacterial origin 's；Extra (external) DNA can easily be received, and can be easily introduced in suitable host cell.

The Shigella of the present invention preferably comprises the restructuring endogenous invasion and attack plasmid as carrier to express one or more Heterologous gene.Therefore, according to a preferred embodiment, which is " prosthetic carrier " bacterial strain, means, removes Beyond any (restructuring) endogenous plasmid, which does not include artificial plasmid.

Plasmid vector generally comprises coding DNA and promoter DNA, and with one or more is suitable for being inserted into external The restriction site of DNA.Coding DNA is the DNA sequence dna of the specific amino acid sequence of coding specific protein or enzyme.Promoter DNA It is the DNA sequence dna of startup, adjusting or mediation or control coding DNA expression.Promoter DNA and coding DNA can come from phase Same gene or different genes, can be from identical or different biology.Recombinant cloning vector generally includes one or more For dubbing system, the one or more label for being used to screen in host cloned or expressed, such as antibiotic resistance, and One or more box genes.Term " expression system " refers to the host cell being under appropraite condition and compatible carrier, example Such as it is used for the albumen for expressing the foreign DNA coding for being carried by carrier and being introduced host cell.

Therefore, attenuated shigella of the invention is particularly used in the exploitation of live vaccine.

Attenuated shigella is especially obtained from the pathogenic strain of any shiga's strain and sero-group (serotype).For example, Any following group：

Serogroups A：Shigella dysenteriae (12 kinds of serotype)

Serogroup B：Shigella flexneri (15 kinds of serotypes and blood serum subtype)

Serogroup C：Shigella boydii (18 kinds of serotype)

Sero-group D：Sonnei shigella dysenteriae (a kind of serotype)

The pathogenic Shigella bacterial strain used herein for attenuation purpose can be pathogenic strain known to clinic or identification For the bacterial strain comprising virulence factor.Particularly, which is selected from any：Shigella flexneri, sonnei shigella dysenteriae, dysentery will are congratulated Bacterium and shigella boydii, particularly Shigella flexneri 2a, such as Shigella flexneri 2a 2457T (ATCC 700930, DNA= 700930D-5), or CIP 107659 (Institute Pasteur, France).

The Shigella bacterial strain that causes a disease is modified by methods known in the art, including multiple continuous passage, temperature are quick Sense attenuation, mutation etc. so that obtained strains are attenuated, particularly nontoxic, it is impossible to cause main body diseased.

In some embodiments, the modification to pathogenic strain causes the deletion and/or passivation of gene, including reduces or press down The expression of the polynucleotides or gene of system coding virulence factor, or cause the expression of non-functional virulence factor.

There are many known technologies in this area to obtain Attenuating mutations, such as reducing or eliminating polynucleotides expression. For example, using recombinant DNA technology, mutation can be incorporated into predetermined site, such as promoter region or coded sequence to produce nothing Justice mutation.Recombinant DNA technology includes cloned target gene, by rite-directed mutagenesis modifier sequence, limits enzymic digestion, Ran Houchong New connection, then substitutes wild type gene with mutator.

Suitable standard recombinant dna technology is known in the art, and is particularly described in Sambrook et al., “Molecular Cloning:A Laboratory Manual”(1989),2nd Edition(Cold Spring Harbor Laboratory press)。

Attenuating mutations can use method well known in the art to carry out, including the DNA sequence dna of wild type gene is cloned into load Selected marker is alternatively inserted into the DNA sequence dna of clone or deleted partial dna sequence for example in plasmid, cause its blunt by body Change.The introducing of deletion can be for example, by with restriction enzyme cutting DNA sequence, and the restriction enzyme is in coded sequence or only on the outside of it Two points at cut, then two ends of remaining sequence are linked together.Alternatively, mutation allele can be built, its The flank region of middle target gene is individually expanded and is directly linked together in the reaction of single over-lap PCR, without therebetween Target sequence.Carrying the plasmid of the DNA sequence dna of mutation can be shifted by known technology such as electroporation chemical conversion or connection Into bacterium.Then mutant can be identified by suitably selecting, wherein by homologous recombination, the DNA sequence dna of passivation recombinates Into the chromosome of bacterium, wild-type DNA-sequence is endowed non-functional.

In addition, if using antibiotics resistance gene, usually it is removed from bacterium before they are used for vaccine.Root According to the method for Datsenko et al. (Proc.Natl.Acad.Sci.U.S.A 97,6640-6645 (2000)), mutation is to be based on λ Bacteriophage Red recombination systems, the system can specifically destroy plasmid-encoded and chromosome coding gene.The strategy is example Such as substitute these genes with selective antibiotic resistant gene, the selective antibiotic resistant gene is used by PCR and mesh The primer that mark gene has the homologous extensions of 40-60nt produces.These homologous sequences mediate the restructuring based on Red.After selection, antibiotic Also the assistant carrier of expression FLP recombinases can be used to eliminate for resistant gene, and FLP recombinase uses are connected on antibiotics resistance gene The FRT of both sides is directly repeated (FLP identifies target).

In some embodiments, via insertion, delete, or with another nucleotide substitute a nucleotide for example put it is prominent Become, mutation can introduce the predetermined site of chromosome or exchromosomal DNA such as plasmid, it causes expression reduction or prominent without expression Become gene.The mutation should produce the Shigella bacterial strain for the reduced capability for causing dysentery.Preferably, mutation is to delete mutation, its The destruction of middle gene is caused by the shearing of nucleic acid.This mutation can be realized for example by deleting the base-pair of adjacent span.Very Extension to very small such as 10 base-pairs of deletion can cause gene not encoding proteins or coding non-functional albumen.Even The deletion of one single base pair can cause no albumen or non-functional albumen because this mutation as a result, other base-pairs No longer in correct reading frame, or transcription is suppressed or eliminates.It is highly preferred that longer extension is removed such as 100 Base-pair or extremely oligogenic major part, such as at least the 50% of gene.Even further preferably, delete whole gene.

Compared with the mutation of classics induction, the mutation that explicitly defines and manufacture intentionally (including fragment or whole gene are deleted Remove, or combinations thereof) have an advantage in that they do not return to wild type.Therefore, in some embodiment party of the present invention In case, vaccine strains include Shigella Attenuated strain, wherein the mutation of the gene of coding virulence factor includes deleting or inserts Enter to destroy the polynucleotide sequence of coding virulence factor so that no corresponding albumen produces, or albumen is non-functional.

The exemplary virulence factor for selecting to transform attenuated shigella bacterial strain is rfb, ipaB, ipaC or aroC.

Attenuation can be for example by deleting and/or be passivated one or more following genes, or its (big) part, or any causes The regulator for the gene that the gene weakens and bring：Rfb, aroA, aroC, aroD, aroE, virG and ipaA-D.This It is the dual or multiple mutation bacterial strain with least three or more Attenuating mutations to invent preferable attenuated shigella bacterial strain. Restructuring for the preferable target gene of Attenuating mutations includes at least one rfb genes (such as rfb F, D, C, E, J and/or I Gene) and at least one ipa genes (such as ipaB, ipaC).

As the replacement by the obtained Attenuating mutations of genetic modification, the naturally occurring bacterium of Shigella can also be identified Strain, the bacterial strain are nontoxic or dash forward in the polynucleotides or gene of coding virulence factor comprising one or more pre-existing Become, it can be used as live vaccine strain.The Shigella bacterial strain of these naturally occurring, once separated by standard technique, can Further mutation or recombinant DNA technology are undergone to build dual or multiple mutation bacterial strain.

The technology known to those skilled in the art for being used to identify bacterium, the bacterium have in the gene of coding virulence factor There are one or more mutation.Therefore, the routine techniques for the Shigella bacterial strain being mutated for detection by technology described above Northern and western blot method, PCR, ELISA and the cell toxicity test described including the place beyond herein.Without volume The mutant strain of the functioning gene of the specific virulence factor of code can be readily selected out using standard technique.

The gene for encoding virulence factor to be weakened can be that plasmid carries.Therefore, in some embodiments, to causing The modification of case record Hayes bacteria strain includes the one or more endogenous Shigella plasmids of mutation.Term " plasmid " especially refers to solely Stand on the cytoplasmic DNA of bacterial chromosome duplication.The mutation that plasmid was caused a disease or attacked to part Shigella can be designed, is even disappeared Except the mutation of plasmid.However, it is preferred to attenuated shigella more preferably stablizes invasion and attack plasmid still comprising endogenous invasion and attack plasmid. This ensures the stability of attenuated strain, is especially considering that attenuated cell causes the potential forfeiture of invasion and attack plasmid, or potential intake Obtained from (natural) the invasion and attack plasmid of wild type Shigella, these situations can occur for unstable bacterial strain or unstable invasion and attack plasmid.

Shigella invasion and attack plasmid is endogenic in most of Shigella bacterial strains.Although plasmid is attacked in culture will May be lost during Hayes bacteria strain, but it engineered can show the recombinant plasmid of high stability to obtain, make its into To develop useful carrier with the attractive target of the heterologous gene comprising coding for antigens particularly protective antigens.

The feature that there is the restructuring invasion and attack plasmid of the invention obtained in example described below Fig. 5 and 10A to show.

The present invention plasmid include its in Shigella can autonomous replication derivative.This derivative can be corresponding In the derivative of a part for invasion and attack plasmid, the part rather than the region for being responsible for attacking are removed from, or can be corresponding In the derivative of a part for invasion and attack plasmid, which is inserted into by another (heterologous) DNA sequence dna.So as to obtain in shiga Strain can autonomous replication suitable shuttle vector.This carrier can use in any Shigella attenuated live vaccine bacterial strain of the present invention In, or used in can include invasion and attack plasmid any other bacterium include enteroinvasive E.Coli in.

Endogenous Shigella invasion and attack plasmid is fully characterized in this area, and this knowledge is given in these matter The selection in site recombinated in grain and the information of suitable Reproduction Conditions, such as ntds between IS100 and ipaJ genes (these positions are determined for the pCP301 invasion and attack matter of 301 bacterial strains of Shigella flexneri 2a for position between 103187-103328 Grain).

Plasmid is preferably employed as single copy plasmid.

The plasmid of the present invention can provide in a separate form, such as prepare the common of Plasmid DNA by using from cell Method prepares cytoplasmic DNA fragmentation from Shigella cell.In addition, DNA fragmentation can pass through density-gradient centrifugation method, agarose Gel electrophoresis etc. purifies.

For the structure of shuttle vector, whole sequence or one part can be used.When using one part, this part leads to The region of responsible plasmid replication is often included, but can exclude to replicate nonessential region.For example, the definite of region needed for replicating can As obtained by by restriction enzyme digested plasmid be attached partially to can in Shigella autonomous replication plasmid, with the restructuring of acquisition Plasmid converts another bacterium, such as another Shigella bacterial strain or coli strain, and determines whether transformant is heavy comprising this Group plasmid.

The known technology for preparing attenuated bacteria vaccine can be used to prepare for the vaccine of the present invention.Vaccine is advantageously in current In oral, such as aqueous solution or the dry powder for being reconstructed before administration in suitable buffer solution.Advantageously in suitable pH Buffer solution in reconstruct, to ensure the vigor of bacterium.In order to protect attenuated bacteria or vaccine to be destroyed from stomach acidity, give every time Protective agent such as sodium acid carbonate is advantageously given during vaccine.Alternatively, vaccine is presented with lyophilized packing forms.

Vaccine strains can be pharmaceutically given in acceptable instrument, such as spraying or are mixed in food and/or water Or delivered with the mixture with suitable carrier, diluent, adjuvant or excipient such as sterile water, physiological saline, glucose. Given and preparation approach according to required, the material that vaccine strains can include auxiliary for example soak or emulsifying agent, pH buffer, assistant Agent, gelling or adhesion promoting additive, preservative, flavor enhancement, colouring agent etc..It is used to prepare the pharmaceutical carrier of pharmaceutical composition and medicine It is well-known in the art, such as " the Remington's Pharmaceutical Sciences " gone out as given in textbook (1990),18th Edition(Mack Publishing Co.)。

The vaccine strains of the present invention can be examined with multiple dose and by technology known to the technical staff of medicine or veterinary applications Consider such as age, gender, weight, the situation of species and acceptance subject and give the factor of approach and given.The approach of giving can To be cutaneous routes, give via mucous membrane (for example, oral, nose, anus, vagina) or via parental routes (it is intracutaneous, it is intramuscular, Subcutaneously, intravenously, or in peritonaeum).Vaccine strains can individually be given, or can with it is other treatment therapy give altogether or successively to Give.The form given may include suspension, syrup or elixir, and give for parenteral, subcutaneous, intracutaneous, intramuscular or intravenously Give prepared product such as sterile suspension or lotion of (such as injectable is given).

Vaccine can be used for main body vaccine inoculation, particularly the mankind or warm-blooded mammals, especially including pig.

Once producing, vaccine strains of the invention can give main body during active immunotherapy, especially by epidemic disease Seedling is inoculated with, to prevent intestines problem, particularly as Shigella and alternatively caused by heterologous enteron aisle or diarrhoeal diseases substance Dysentery.This can be reached by any vaccine of intersecting protective of the present invention and/or multivalence.

By Shigella and alternatively other microorganisms intersecting protective for example of the present invention and/or polyvaccine targeting Therefore infection caused by diarrhea microorganism can be prevented or be treated by giving a effective amount of vaccine of the present invention.The dosage of use It is finally at discretion by doctor, but include the size and weight of main body and the class of the vaccine of preparation dependent on different factors Type.It is but oral comprising 10⁷-10¹¹Such as 10⁸-10¹⁰Every dose of bacterium is convenient to 70kg adult human hosts.

Alternatively infecting as Shigella and caused by other microorganism particularly pathogen can be therefore by having given The vaccine of the present invention of effect dosage is prevented or is treated.The dosage of use is finally at discretion by doctor, but dependent on different Factor includes the size and weight of main body and the type of the vaccine of preparation.It is but oral comprising 10⁷-10¹¹Such as 10⁸-10¹⁰ Every dose of bacterium is convenient to 70kg adult human hosts.

According to specific example, construct prototype Shigella flexneri 2a bacterial strains waits gene attenuated mutants.Mutant or Person cannot synthesize O antigens (Δ rfbF), or-representing the vaccine approach fully proved-is auxotrophy (Δ aroC).These Pathogenic display comparable levels of attenuation of the mutant in Mouse Lung Model.Then, we, which isolate, lacks coding Ipa-s's Attack the derivative (Congo red feminine gender/CRN/ mutant) of two kinds of mutant of plasmid.The mutant loss invasion and attack of these latter Attenuation is further increased to undetectable level by phenotype.Lack O antigens (Δ rfbF CRP) or ipa albumen (Δ aroC CRN) Or both (Δ rfbF CRN) or this Shigella flexneri 2a mutant series without these antigens (Δ aroC CRP) be used for Sublethal dose is through intranasal vaccinated mice.Then, congratulated with the heterologous Shigella flexneri 6 (Fig. 1 a) or sonnei will of lethal dose Bacterium (Fig. 1 b) wild-type strain attacks mouse.The attenuated mutants of expression ipa and O antigens (Δ aroC CRP) cannot be provided to mould Intend the horizontal protection observed of vaccine inoculation mouse.On the contrary, lack the double mutations of two kinds of main immunogenic antigen groups Body causes the height for heterologous sexual assault bacterial strain to be protected.The plasmid (Δ aroC CRN) that causes a disease even only is lost to seem all improve Cross protection.In order to confirm these as a result, each group mouse also uses the I phases (Ipa and O antigen positives) of sonnei shigella dysenteriae isolate It is immunized with II phases (Ipa and O antigen negatives) variation.II phases vaccine strains provide the protection attacked Shigella flexneri 6, and The offer survival rate that there was no significant difference compared with brine is immunized with completely pathogenic I phase bacterial strains.

In order to support that under this mutant background it is secondary that (lose main antigen-see Fig. 2) improved cross protection is derived from The theory of the immunity of antigen enhancing, will be obtained from receiving the serum and bronchoalveolar lavage (BAL) sample of immune mouse Immunoreactivity in ELISA expression main antigenic group both or do not express and contrasted on its whole bacterial cell (Fig. 3).

BAL obtained from the mouse that vaccine inoculation is carried out with Δ aroC CRP (ipa and O antigens are the positive) mutant is to invading The smooth homology target of attacking property has more reactionogenicity, it was demonstrated that these antigens dominate immune response really.On the contrary, vaccine strains (Δ RfbF CRN) on lose the reaction of the homology aimed strain that immune leading antigen causes to lacking both O antigens and Ipa antigens Originality increase.

In addition, heterologous sonnei shigella dysenteriae bacterial strain is easier by obtained from the mouse that vaccine inoculation is carried out with double mutant BAL identification.These results demonstrate that for those come-at-able shared minor antigens in these targets mucoantibodies have compared with High titre.It is interesting that this phenomenon in the case of serum IgG and unobvious (data are not shown), supports the hair of early stage Existing, the discovery of early stage is shown, sIgA rather than the serum IgG mediate protection in this model.

Current vaccine approach (in general also referring to particularly Shigella) relies on the utilization of main immunogenic antigen. But in order to avoid immune response, evolution pressure has selected the upper different variation of the panimmunity of these antigens, this results in The basis that pathogen is classified according to serotype.Therefore, can only be assigned for pathogen using the major antigen of definite serotype Part protection, except not all serotype may include in vaccine (such as in the case of poliovirus vaccine).Most The combination of popular serotype can give wide protection, and still, since serotype is replaced, (i.e. more uncommon serotype goes out It is existing, fill the notch that the elimination of vaccine serotype is opened), this is probably of short duration.This makes it necessary to optimize epidemic disease from time to time Seedling, such as by the way that extra serotype is covered in polyvaccine.However, due to such as antigenic competition and the phenomenon of interference with And economic consideration, the maximum quantity of serotype to be covered are restricted.

On the other hand, the different serotypes of set bacterial pathogens share substantial amounts of conservative antigen on its surface.They are still So kept for the fact that conservative imply, their (non-protective antigens) non-accessible on the surface and/or their function are for hair Sick so indispensable, its antigenic structure does not allow to modify.This is guarded by Shigella ipa albumen illustrations, the albumen height (due to their sophisticated functions in terms of invasion and attack) and there is suitable immunogenicity, it cannot still cause cross protection, have very much It is probably because they are only expressed in contact target cell, therefore probably cannot be used for antibody-mediated protectiveness machine System.

Particularly, we show (Fig. 2), and immune leading antigen such as Iipa and O antigens are next to allow less antibody rise The mode of confrontation minor antigen kidnaps immune response.In view of Ipa-s is non-protective and O antigen alterable heights, shiga Bacterium can effectively avoid immune response.But we show, the deletion of these classification antigens has been highly improved the friendship of live vaccine strain Fork protection potentiality.

As for invasion and attack plasmid encoding mutant, the spontaneous deletion mutant except attacking plasmid based on Congo red positive forfeiture selection, Also removable ipaB and C genes, while plasmid remainder keeps complete.

In addition, can be by transplanting essential gene such as ppa to invasion and attack plasmid from chromosome come stable plasmid.

In addition, the expression of external (heterologous) antigen such as ETEC LTB and STa (STm) toxin of mutation are feasible. STm preferably comprises one or more point mutation.Particularly preferred STm is

NSSNYCCELCCXXACTGCY (SEQ ID 1),

Wherein

X at position 12 is N, K or R, and/or

X at position 13 is P, G, L or F,

Wherein STm does not include wild-type sequence：

NSSNYCCELCCNPACTGCY(SEQ ID 2)。

Preferable point mutation combination is the combination of N12K or N12R and P13F.

In addition, example is shown, the comparative immunogenicity of shared conservative antigen is raised when vaccine strains lack major antigen. Since these deletions not only improve protection domain, vaccine strains height nontoxicity is also assigned, therefore double mutant is considered as It is very safe, or even be also in high dosage.In addition, the manufacture of live oral vaccine is relatively inexpensive, and need not be trained Medical worker give, this is important factor when considering popular national objective colony.

Theme defined below is considered as embodiment of the present invention：

1. Shigella attenuated live vaccine, it is based on the coarse Shigella for lacking LPS O antigens, preferably Non-Invasive bacterium Strain.

2. the vaccine according to defining 1, it is attenuated by following mutation：One included in LPS synthesis, transhipment and expression One or more in the rfb/wbb gene clusters of a or multiple genes, the gene being preferably selected from rfb clusters or coding O antigen synthesis A gene, encodes the waaL of O antigen ligases, encodes the wzx of the O antigen flippases included in O antigen deliveries, the polymerization of O antigens In the wzy/rfc that includes, the gene in the rfa/waa gene clusters of coding LPS core synthesis, influences the adjusting bases of O antigen presentations Cause, such as rfaH, or missing cause the function of O antigen presentations reduction at least 90%.

3. the vaccine according to defining 1 or 2, wherein the mutation is by deleting rfb F, D, C, E, J and/or I gene One or more of, or one part is deleted, or delete corresponding gene in different Shigella serotypes.

4. according to the vaccine for defining any one of 1-3, wherein the Shigella bacterial strain is selected from Shigella, it is selected from Any Shigella serotype or kind, particularly Shigella flexneri, sonnei shigella dysenteriae, Shigella dysenteriae and shigella boydii.

5. according to the vaccine for defining any one of 1 to 4, wherein the Shigella expresses cross reactivity outer membrane protein.

6. according to the vaccine for defining any one of 1-5, its Shigella of the same race to different serotypes and not, particularly to good fortune Family name's shigella dysenteriae 2a, Shigella flexneri 6 and sonnei shigella dysenteriae or enteroinvasive E.Coli are one of any to have intersecting protective.

7. according to the vaccine for defining any one of 1-6, wherein the Shigella is special by being further mutated invasion and attack plasmid It is that deletion ipaB and/or ipaC genes and/or other ipa genes become no invasion.

8. according to the vaccine for defining any one of 1-7, wherein the Shigella includes restructuring endogenous invasion and attack plasmid, it is described Plasmid includes at least one gene of encoding heterologous antigens, to secrete the antigen or by the antigen presentation in such as bacterium Property cell surface.

9. according to the vaccine for defining 8, wherein the antigen is selected from：

- bacterial antigen, preferably toxin or colonizing factor,

- viral antigen, is preferred from the cause of disease for causing enteron aisle or mucosal infections,

- fungal allergen, is preferred from the cause of disease for causing enteron aisle or mucosal infections, and

- parasitics antigen, is preferred from the cause of disease for causing enteric infection.

10. according to the vaccine for defining 9, wherein the bacterial antigen is derived from enteropathic bacteria, it is preferably selected from：

A. bacillus coli antigen, is especially selected from following enterotoxin：The LTB of ETEC, the LTA and ST, subunit of mutation, Or its fusion, the antigen from enteroinvasive E.Coli (EAEC), or shiga-like toxin 1 or 2

B. campylobacter jejuni antigen,

C. clostridium difficile antigen, particularly toxin A and B

D. comma bacillus antigen, particularly CT-B antigens, and

E.a), mutant or fusion protein b), c) or d).

11. according to the vaccine for defining 10, wherein the ETEC is the fusion enterotoxin of LTB and mutation ST (STm), particularly Include the fusion protein of the STm with the amino acid sequence as shown in SEQ ID 1.

12. according to the vaccine for defining 10, wherein the viral antigen is derived from diarrhea virus, be preferably selected from rotavirus with Calicivirus, such as norwalk virus.

13. according to the vaccine for defining 10, wherein parasitics antigen comes from the protozoan for causing diarrhea, is preferably selected from merchant the Flagellate, the kind of Cryptosporidium and Entamoeba histolytica.

14. according to the vaccine for defining 10, wherein fungal allergen is derived from the fungi for causing diarrhea, is preferably selected from the life of dermatitis bud Bacterium and Histoplasma.

15. according to the vaccine for defining any one of 1-14, wherein the Shigella further comprises deleting necessary chromosome Gene, and the gene is inserted into invasion and attack plasmid, particularly ppa genes, or accD, acpS, dapE, era, frr, FtsI, ftsL, ftsN, ftsZ, infA, lgt, lpxC, msbA, murA, murI, nadE, parC, proS, pyrB, rpsB, Any one in trmA, rho and rhoL.

16. according to the vaccine for defining any one of 1-15, for the active immunotherapy of main body to prevent from catching, especially It is intestines problem such as diarrhea or diarrhoeal illness.

17. according to 16 vaccines used are defined, wherein the intestines problem is caused by any Shigella serotype or kind.

18. according to 16 or 17 vaccines used are defined, wherein immunotherapy includes giving vaccine with mucous membrane or oral formulations.

19. the vaccine used according to any one of 16-18 is defined, wherein being given in the vaccine is oral or nasal.

20. the vaccine used according to any one of 16-19 is defined, wherein

- polyvaccine is used for expressing the protection of at least one pathogen of Shigella and the kind in addition to Shigella Property antigen, it is attacked in plasmid by bringing into the protective antigens of the cause of disease through endogenous, and wherein

- the infectious diseases is caused by any Shigella serotype or kind and/or the pathogen.

21. Shigella bacterial strain, it is to delete rfbF, ipaB and/or ipaC genes, or deletes its necessary part Shigella flexneri 2a bacterial strains.

22. according to the Shigella bacterial strain for defining 21, it includes a restructuring invasion and attack plasmid, and the plasmid includes at least one The gene of encoding heterologous antigens is to express the antigen or the secretion antigen.

23. according to the Shigella bacterial strain for defining 21 or 22, it further comprises deleting necessary chromogene and insertion The gene is into invasion and attack plasmid.

24. the recombinant plasmid vector of the Shigella invasion and attack plasmid based on mutation, it is at least one that the plasmid includes coding The nucleotide sequence of heterogenetic antigen, wherein the plasmid is mutated at least one ipaB and/or ipaC genes.

25. include the bacillary host cell for the carrier for defining 24, wherein the host cell is selected from Shigella, big Enterobacteria, salmonella, campylobacter or Yersinia.

26. according to the host cell for defining 25, wherein the carrier is endogenous invasion and attack plasmid.

Described above can be more fully understood by by reference to following examples.But these example is only to represent this One or more implementations of invention, and should not be construed as limiting the scope of the invention.

Example

Example 1：The preparation of Shigella bacterial strain

Method

Bacterium bacterial strain and condition of culture

Bacterium routine growth is in Luria Bertani (LB) meat soup or on agar plate.In order to detect expression ipa albumen Complete invasion and attack plasmid, use the Tryptic Soy Agar for being supplemented with 0.01% congo red (Sigma-Aldrich) (TSA) tablet.Since fresh culture always ensuring to carry Congo red positive (CRP) bacterium colony of plasmid.On appropriate ground Side, with the antibiotic supplementing culture medium of following concentration：Ampicillin 100 μ g/ml, kanamycins 100ug/ml, 25 μ of chloramphenicol g/ml。

Parenteral is used as using the prototype shigella flexneri 2a bacterial strains 2 (bacterial strain 2457T, ATCC 700930) through sequencing Bacterial strain is used to be mutated.The passivation of aroC and rfbF genes by the Red that describes before recombinate zymotechnic carry out (Levine, M.M., et al.Nat.Rev.Microbiol.5,540-553(2007)).The deletion of aroC causes that aromatic compound cannot be synthesized Auxotrophic mutant.RfbF is included in the synthesis of O antigen subunits, its forfeiture can cause coarse LPS phenotypes.For producing Oligonucleotides that is raw and confirming mutant provides (SEQ ID 3-10) as the supplementary table 1 in Fig. 4.Then by mutant 2457T ΔaroC::Kan and 2457T Δs rfbF::Cat is cultivated on Congo red (CR) agar plate to select CR positive (CRP) and CR Negative (CRN) bacterium colony.The forfeiture of the pathogenic determinant encoded on invasion and attack plasmid is confirmed by PCR.Similarly, sonnei will The I phases and II phases variation for congratulating bacterium distinguish on CR tablets.The I phases are the biographies of the wild type sonnei bacterial strain comprising invasion and attack plasmid System design, and the II phases refer to plasmid deletion mycopremna.Since the synthesis of O antigens is also encoded on the pathogenic plasmid of this species, II Phase variation is both Non-Invasive, and coarse.Wild type Shigella flexneri 6 and sonnei shigella dysenteriae for Attack Research Bacterial strain is separated from the clinical case of bacillary dysentery.Their serotype by using business blood grouping serum slide Agglutination determines.

Zoopery

All In vivo studies carry out (van de Verg et al., Infect in the Mouse Lung Model described before Immun63:1947-1954,1995).6-8 week old female BAl BIc/c mouse 5mg/ml ketamines (Calipsol, Richter Gedeon, Hungary) and 0.3mg/ml xylazines (Primasine, Alfasan) mixture intraperitoneal anesthesia.

Infected by using the inoculum (being diluted in brine) of the 50 μ l bacteriums for including required CFU through intranasal.Pass through Spread plate is serially diluted with inoculum, it was demonstrated that count of bacteria is rational.According to Reed and Muench (Oaks, EV, et al.Infect.Immun.53:57-63,1986) (10 are serially diluted with 0.5log-⁵-10⁸CFU) infection carried out calculates 50% Lethal dose (LD50 values).With the bacterium (10 from bacterial strain 2457T of sublethal dose⁶The CRP mutant of CFU and 10⁸Cfu's CRN mutant；10^5.5The I phases of CFU and 10^7.5The II phase sonneis shigella dysenteriae of CFU) carry out vaccine inoculation twice, interval is 2 weeks, Complete vaccine inoculation.Control group receives brine.In Primary Study, it has been suggested that all vaccine strains are removed in 3 days after injection. Two weeks after enhancing is immune, mouse is attacked with the Shigella flexneri 6 or sonnei shigella dysenteriae wild-type strain of lethal dose.

Then, lethal is monitored 14 days.Alternatively, immunized mouse puts to death and collects bronchovesicular for two weeks after enhancing Lavation (BAL) liquid and blood sample.Collection for BAL, prepares the tracheae of euthanasia mouse, is intubated with blunt needle and injects 200 μ l salt Water, from the bronchus extraction of every mouse.

ELISA

It is inoculated with from the bacterium of fresh CR tablets and is grown in LB meat soups overnight.96 orifice plates (C.E.B., France) use 0.1ml Washed bacterial suspension (5x10 in carbonate buffer solution (pH 9.5)⁸CFU/ml) coated overnight at 4 DEG C.Second My god, with the PBS cleaning tablet comprising 0.05% polysorbas20, then with the PBS comprising 2%BSA (Sigma-Aldrich) in room temperature Lower closing 1h.BAL and serum samples diluted are incubated in the PBS comprising 0.5%BSA with the tablet of antigen coat at 37 DEG C 1h.It is serially diluted on tablet.After cleaning three times, with the anti-mouse IgG connected with HRPO (Dako A/S, Denmark) (being used for serum IgG) or anti-mouse IgA (being used for BAL samples) Detection of immuno-protein tablet.ELISA substrates, which are dissolved in, to be included H₂O₂Citrate buffer solution in o-phenylenediamine (Sigma-Aldrich).In conventional ELISA plate readers at 492nm Measure OD.Immunoreactivity is expressed as in identical dilution factor (1:10) reactivity of Δ aro CRP BAL samples under.From 4 times solely Vertical experiment calculation average+SEM.

Statistical analysis

50% lethal dose is calculated with the statistical method of Reed and Muench 6.The statistics credit of Survival curves Analysis is examined with LogRank (Mantel-Cox) and carried out using the GraphPad Prism version 5.00 for Windows. The IgA titres of BAL are compared with the analysis of Mann-Whitney printenvs.If p value is less than 0.05, it is considered as significantly.

As a result

Based on being immunized with attenuation Shigella flexneri 2457T (serotype 2a) vaccine strains and used wild type Shigella bacterial strain The Survival curves of the animal of attack, the forfeiture of plasmid is attacked by combining in rfbF gene elminations and heterologous sexual assault setting, It was observed that coordinating protection effect.When with 6 strain challenge animal of heterologous Shigella flexneri, with Congo red negative (CNR, elimination Invasion and attack plasmid) Shigella flexneri 2457T (2a) Δ rfbF bacterial strains be immunized relative to it is Congo red the positive (CNP, is completely invaded Attack plasmid) Shigella flexneri 2a Δ rfbF bacterial strains carry out immune obtaining significantly preferable protection (Fig. 1 b).With wild type Fu Shi In the case that 544 bacterial strain of shigella dysenteriae (2a) carries out homologous sexual assault, two kinds of vaccine strains have equal protectiveness, prompt for same Source is attacked, even if it is all enough (Fig. 1 a) to delete rfbF genes.The difference of vaccine inoculation effect is likely to allow with higher The Δ rfbF- invasion and attack plasmid double mutations body portions of sub- lethal challenge dosage are related, but endless total correlation, because to dash forward with dual CRN Δs aro (control) bacterial strains that the suitable challenge dose of variation uses cause part in homology and heterologous sexual assault experiment Protect (Fig. 1 a, b).

It is passivated rfbF genes and attacks plasmid encoding mutant acquisition for the beneficial effect significantly protected of heterologous sexual assault Further evidence using sonnei shigella dysenteriae vaccine strains by being provided.(responsible table is deleted with sonnei shigella dysenteriae II phases variation Up to the invasion and attack plasmid of the expression of invasion and attack complex and both rfbF genes) immune provide for wild type Shigella flexneri 542 The high level protection of the lethal hit of bacterial strain (serotype 6), and wild type sonnei shigella dysenteriae I phases variation (it is multiple to carry invasion and attack Fit and rfbF genes complete invasion and attack plasmid) low (statistically not notable) protecting effect (Fig. 1 c) of display.

Example 2：The preparation of 2457 mutant of Shigella flexneri 2a with synthetic gene construction is attacked on plasmid

The source material of mutant construction is ATCC bacterial strains Shigella flexneri 2a 2457T as described above.RfbF and ipaB and The deletion of ipaC genes and ppa genes carries out (Datsenko, K.A.＆Wanner, B.L.One- using Red restructuring zymotechnics step inactivation of chromosomal genes in Escherichia coli K-12using PCR products.Proc.Natl.Acad.Sci.U.S.A 97,6640-6645(2000))。

Step 1：RfbF genes are removed from chromosome.The missing of RfbF is related to phenotypic alternation：Shigella bacterial strain Become " coarse ", it is a kind of to be changed by the representative configuration that naked eyes detect on agar plate.This phenotypic alternation is seen Observe, but the successful removal of rfbF genes is also confirmed by PCR analyses.This is the shiga based on wild type or mutation The length for the PCR product that the genomic DNA of bacterium obtains is different (Fig. 8).

Step 2：IpaB and ipaC genes are removed from invasion and attack plasmid.These genes are adjacent, and are applied to identical The Red restructuring zymotechnics of rfbF gene elminations are deleted together.This gene elmination also results in a phenotype：Shigella loses intake Therefore the Congo red ability of dyestuff simultaneously forms white colony on comprising Congo red agar plate, with carrying wild plasmid The red that Shigella is formed is contrasted.Due in Shigella in vitro incubation can spontaneous loss its plasmid, The deletion of ipaB and ipaC genes analyzes mutant to confirm by PCR, and it is based on compared with wild plasmid, mutant The PCR fragment of acquisition is shorter (Fig. 9).

Step 3：The insertion of the synthetic gene of ETEC toxin LT-B and ST expression is driven, and necessary base is transplanted from chromosome Cause (ppa) is into invasion and attack plasmid (referring to Figure 10 A).The position being successfully introduced into by genetic manipulation region of LTB-mST fusions Point specific PCR is expanded to prove (Figure 10 B).The expression of toxin fusion gene from Shigella is surveyed by Western blot Try (Figure 10 C).Ppa genes (growth to Shigella is necessary) are based on from final from the removal of chromosome by PCR The amplified production length of vaccine strains is shorter to be proved.

All genetic manipulations include the insertion of antibiotics resistance gene.After each step, it is responsible for antibiotic resistance Gene remove (Proc.Natl.Acad.Sci.U.S.A with such as described helper plasmids of Datsenko and Wanner 97,6640-6645(2000))。

Example 3：The animal protection research of the mutant strain of test case 2

The mouse lung of shigellosis is shown in the attenuation of their parent wild type strain Deng gene mutation bacterial strain In model.Each group mouse is serially diluted (10 with 10 times of different bacterium bacterial strain⁶To 10⁸Between cfu) by intranasal infection to determine The minimal lethal dose of every kind of bacterial strain.In the case of wild-type strain, 10⁶, 10⁷With 10⁸Sent out respectively under cfu/ mouse doses Existing 30,50 and 100% lethal.On the contrary, wait gene mutation body 2457T Δs rfb, 2457T Δ ipaBC or double mutant Any one of 2457T Δ rfb Δs ipaBC does not have dead mouse under any proof load.These results are prompted, and are deleting phase All mutant are highly attenuated when answering gene.

Then, each group mouse uses the wild-type strain 2457T (5 × 10 of sublethal dose in identical model⁵Cfu/ is small Mouse) or its grade gene elmination mutant 2457T Δs rfb, 2457T Δ ipaBC, 2457T Δ rfb Δ ipaBC it is (all to be 10⁸Cfu/ mouse) it is immune, or only simulated with PBS immune.Carry out identical immune three times, interval is 2 weeks.Last time strengthens Latter week, with the lethal dose (2 × 10 of (optimizing) sonnei shigella dysenteriae bacterial strain before⁶Cfu/ mouse) attack.Such as Figure 11 institutes Describe, protection cannot be provided by with wild-type strain be immunized, and each single locus mutation (2457T Δs rfb or 2457T Δ ipaBC) only cause part to be protected.Congratulated on the contrary, double mutant (2457T Δ rfb Δ ipaBC) can be provided for heterologous will The complete protection of Salmonella kind infection.

Claims (13)

1. Shigella attenuated live vaccine, its Shigella of the same race to different serotypes and not has intersecting protective, described Shigella includes becoming coarse and non-invasion and attack by deleting ipaB and ipaC genes on rfbF genes, and deletion invasion and attack plasmid Property Shigella bacterial strain, wherein the invasion and attack plasmid is restructuring endogenous invasion and attack plasmid, the plasmid includes at least one volume The gene of code heterogenetic antigen, to secrete the antigen or the expression antigen.

2. vaccine according to claim 1, wherein the Shigella bacterial strain is selected from Shigella.

3. vaccine according to claim 2, wherein the Shigella bacterial strain is selected from Shigella flexneri, sonnei shigella dysenteriae, dysentery Disease shigella dysenteriae and shigella boydii.

4. according to the vaccine of any one of claim 1-3, it is to Shigella flexneri 2a, Shigella flexneri 6 and sonnei shigella dysenteriae Or enteroinvasive E.Coli is one of any has intersecting protective.

5. vaccine according to claim 1, wherein the antigen is selected from：

- bacterial antigen,

- viral antigen,

- fungal allergen, and

- parasitics antigen.

6. vaccine according to claim 5, wherein the bacterial antigen is toxin or colonizing factor；The viral antigen comes From the pathogen for causing enteron aisle or mucosal infections；The fungal allergen is from the pathogen for causing enteron aisle or mucosal infections；Institute Parasitics antigen is stated from the pathogen for causing enteric infection.

7. vaccine according to claim 5, wherein the bacterial antigen is an enterotoxin (ETEC), it includes thermally labile poison Plain B subunits (LTB), heat-stable toxin (ST) or subunit or their fusion.

8. vaccine according to claim 7, wherein the bacterial antigen, which includes, has the amino acid sequence as shown in SEQ ID 1 STm LTB/STm.

9. vaccine according to claim 1, wherein the Shigella further comprises deletion and the institute of necessary chromogene Gene is stated to be inserted into invasion and attack plasmid.

10. according to application of the vaccine of any one of claim 1-9 in medicine preparation, the medicine be used for the prevention of main body with Prevent infectious diseases.

11. application according to claim 10, the medicine is used for the prevention of main body to prevent intestines problem.

12. application according to claim 10, wherein being given in the vaccine is oral or nasal.

13. according to claim 10-12 any one of them applications, wherein

The protectiveness at least one pathogen that-polyvaccine is used for expressing Shigella and the kind in addition to Shigella resists Original, it is attacked in plasmid by bringing the protective antigens of the pathogen into endogenous, and wherein

- the infectious diseases is caused by any Shigella serotype or kind and/or the pathogen.